System for analyzing images produced by bacterial reactions

ABSTRACT

It is based on an apparatus ( 1 ) forming a closed chamber ( 2 ) inside of which is installed an upper centered digital camera ( 15 ) oriented toward a removable support ( 17 ) over which is placed either a panel fitted with receptacles for holding the liquid culture mediums or a plate for placing on it a solid culture medium, the support ( 17 ) being located between lower lighting means ( 18 ) and upper lateral lighting means ( 19 ) complemented with light diffusers ( 20 ) and ( 21 ). The apparatus includes external operation indicators, switches, outlets and connections providing for appropriate operation and control through a central computer. The system captures and interprets the images produced by the reactions taking place in said culture mediums, either liquid or solid, allowing the bacterial reactions to be converted into values, in addition to analyzing microorganism growth.

OBJECT OF THE INVENTION

The invention refers to a system capable of converting bacterialreaction images into numerical values and analyzing microorganism growthby capturing images from a panel or plate.

The object of the invention consists in capturing and interpretingimages produced by reactions taking place either in a liquid culturemedium contained inside panel receptacles or on the surface of a solidculture medium in all-purpose laboratory receptacles, or so-calledplates.

BACKGROUND OF THE INVENTION

Liquid culture mediums use support means commonly referred to as panelswhich are usually made of polystyrene, have a rectangular shape and arefitted with receptacles designed to contain various microbiologicalculture mediums, biochemical substrates and/or antimicrobial agents inliquid form in which metabolic reactions occur, the microbiologicalinterpretation of which leads to the identification of a microorganismand/or the definition of its capacity to be neutralized by one orseveral antibiotics.

The cultures may also be performed with the use of another type ofsupport or plates in the form of a container generally made ofpolystyrene, having various shapes and containing a single solid culturemedium on which the bacterial reaction takes place.

Thus, traditionally, in the case of the panels, a visual inspection ofthe reactions occurring the various receptacles is performed in thelaboratory, and, based on the panels' shape and color, the resultprobability is established according to general tables published forthis purpose.

Automatic reading systems are presently used which, by measuring theoptical density of the receptacles, assign a value interpreted aspositive or negative by a computer program. Subsequently, anothercomputer module establishes the result probability by combining thepositive character of the partial reactions.

In the case of the plates, the approximation to the result hastraditionally been more rudimentary in view of the smaller number ofparameters involved, which are based mainly in the amount, size andcolor of the bacterial colonies.

The advantage of the these automatic systems—which measure opticaldensities—over the traditional visual inspection method lies in that iteliminates the user's subjectivity in interpreting reactions, since eachreaction is translated into a numerical value.

However, these automatic systems present a serious problem and drawbackin that they are unable to interpret both the morphological features andthe growth patterns of the bacteria, which thus elude the resultobtaining method of certain important elements, such as the form ofbacterial growth, namely the base for obtaining microbiological resultsduring the past decades.

DESCRIPTION OF THE INVENTION

The proposed system was conceived for the purpose of solving the aboveproblems, allowing microbiological reactions to be converted tonumerical values which are subsequently applied to a diagnosis softwarein order to establish the number of microorganisms present, revealing aninfection, to identify the microorganisms present in the sample or toestablish an organism's antimicrobial susceptibility pattern.

The following reactions are identified via the inventive system:

Bacterial colony size, color, shape and quantity.

Color development in biochemical substrate receptacles, indicative ofthe type of bacterial metabolism involved.

Development of compact shapes or turbidness in antibiotic receptaclesthat would reflect the microorganism's development capability in thepresence of specific antimicrobial agent concentrations.

The formation of a circle around an antibiotic disc, or so-called“inhibiting halo”.

The system is thus based on a device preferably having aprismatic-rectangular shape, the inside of which establishes ahermetically closed chamber of diverse shapes and dimensions providedthey allow for appropriate focal distance between a digital camera lensand the panel or plate to be analyzed. The digital camera is internallymounted on an upper central support, an internal area near the logierend of the chamber being fitted with a support associated to aninlet/outlet feeder arranged for depositing the corresponding panels orplates, i.e. the objects to be analyzed in each case, characteristic inthat the supports are interchangeable and may be of various typesdepending on the panels or plates to be processed.

The function of this support installed inside the chamber, as previouslystated, is to allow the passage of light from the lower part of theobject being processed or analyzed, and to center the object under thedigital camera.

An internal lighting system is also provided, comprising fourfluorescent tubes, two of which are installed along the sides and abovethe object to be processed and the other two are installed under theobject to be processed, so that, depending on the type of image to beprocessed, the upper, lower or both fluorescent lights are activated,this being implemented either automatically, through the functionmanagement software, or manually, by means of a switch.

Externally, the chamber's front panel includes a series of functioncontrol LEDs and switches, in addition to the corresponding feedingmechanisms for inserting the panel or plate to be processed.

The chamber's rear panel includes external outlets leading to a centralcomputer, to a TDM 436 card and a to digital camera and lighting systempower supply line; however, depending on the assembly's operationalrequirements, other outlets may be installed or said outlets modified.

It must also be noted that the rear panel includes an access cover forinternal cleaning, maintenance, component replacement, calibration,etc., purposes.

The system further includes a means of identifying the type of panel andthe external data used for complementing the identification testsperformed in the panel, and a means of establishing the position of eachreceptacle in the panel and calculating the panel area to be used forthe reading.

According to the characteristics thus described, the system, inoperation, must first perform a calibration of the camera by showing acamera histogram and its reading values on the screen of a controlpersonal computer, so that, when an indicator lights up to signal theend of the calibration reading, a panel inserted in the internal supportof the feeder may be read, said panel's upper surface being divided intothree information areas, namely one corresponding to the type of panelidentification area, another corresponding to the external informationarea and a third corresponding to the receptacles area.

Once the corresponding reading option is activated in the controlsoftware, the feeder is closed and an image reading instruction isissued enabling the system-generated data to be interpreted by thesoftware, which assigns the analyzed bacteria a taxonomic classificationand a code as to whether it is resistant or not to a given antibiotic orgroup of antibiotics.

DESCRIPTION OF THE DRAWINGS

In order to complement the description and help toward providing abetter understanding of the characteristics of the invention accordingto a preferred practical embodiment thereof, a set of drawings isattached as an integral part of the description wherein the following isrepresented as illustrative and non-limiting in character:

FIG. 1 shows an external front view of the apparatus or chamber whichforms a part of the system that is the object of the invention.

FIG. 2 shows an external rear view of the same apparatus or chambershown in the preceding figure.

FIG. 3 shows a front view section through a vertical plane of theapparatus or chamber shown in the two preceding figures.

FIG. 4 shows an external side view, also in section through a verticalplane, of the same apparatus or chamber shown in the preceding figure.

FIG. 5 shows a general top view o a panel with three identificationsections corresponding to: type of panel, external information, andposition of each receptacle.

FIG. 6 shows a perspective view of a form of embodiment of a receptaclepanel support.

FIG. 7 shows a further perspective top view of a plate support.

FIG. 8 shows a detail of the manner in which a selection of the readingarea is performed according to the position of each receptacle in thecorresponding panel.

FIG. 9 shows a plan view of a plate including several discs impregnatedby a selection of antibiotics.

Finally, FIG. 10 includes a graph corresponding to the calibrationhistogram.

PREFERRED EMBODIMENT OF THE INVENTION

In the light of the above figures, it is evident that the system of theinvention is based on an apparatus fitted with a general housing (1) ofa prismatic-rectangular shape which internally defines a closed chamber(2), characteristic in that its front panel (3) incorporates a series ofexternal indicator LEDs (4) providing the following functions: indicator(4 a) corresponds to the electric current inlet to in the chamber (2);indicator (4 b) corresponds to the end of the calibration. The threenext indicators (4 c) correspond to 25% 50% and 75%, respectively, ofthe reading performed; indicator (4 d) corresponds to the end of thereading.

Also, said front panel (3) includes a corresponding feeder (5), thefunction of which is described further on, an opening button (6) for thefeeder, a contrast switch (7) and a general switch (8), in a manner thatsaid indicators (4) and elements (5 to 8) constitute the means ofcontrolling system functions and inserting the panel or plate to beread.

The rear panel (9) of apparatus (1) includes a cover (10) providingaccess to the internal components and an electric current outlet (11)for the auxiliary lighting systems; an electric current outlet (12) forthe digital camera described hereunder; a communication port (13) forconnection to the central computer; connections (14) to a TDM 436 modulecontaining the calculating program for converting the image'scalorimetric signals into numeric values, said connections (14)corresponding to blue, green and red colors which provide the signalscorresponding to the percentages of said colors or calorimetric signals.

Inside chamber (2) is fitted said digital camera (15), mounted on anupper support (16), the camera (15) being arranged in a central positionand in line with the center of a support (17) on which the object to beanalyzed is placed, namely the corresponding plate or panel. Support(17) is interchangeable and may vary in shape, depending on the feeder(5) and the type of panel or plate to be supported. In any case, saidsupport (17) is part of the feeding device (5) itself, and constitutesthe means allowing for the insertion of the supports to be processed,i.e. the panels and plates, since pressing the opening button (6)involves sliding the feeder (5) and thus the support (17) guided overrails and activated on its outward movement by any conventional system,from a simple pressure spring to a pneumatic mechanism or similardevice.

Inside chamber (2), in addition to said digital camera (15) and support(17), are included lighting means in the form of a pair of fluorescentlamps (18) located on a lower position and a pair of fluorescent lamps(19) located at an intermediate lateral height, said fluorescent lights(18) and (19) being complemented with respective light diffusers (20)and (21), so that the former lie under the object to be processed,namely under the support (17), whereas the latter lie above it,characteristic in that the latter are complemented with a protectiveplate (19′) forming an inclined metal screen preventing the radiationsto interfere with the readings of the digital camera (15). The lightdiffusers (20) and (21) are preferably made of white crystal and arefitted for providing homogeneous light distribution, in the first caseon the object to be processed and in the second case through thetransparent polystyrene panel or plate receptacles.

Depending on the type of image to be processed, the upper fluorescentlights (19) or the lower fluorescent lights (18), or bothsimultaneously, are activated either automatically, through the softwaremanaging the various functions, or manually, through a contrast switch(7) located on the front panel (3).

FIG. 6 shows a support (17′) for a receptacle panel, which support (17′)is fitted with holes (22) for the panel's receptacles and recesses (23)and (24) for illuminating the panel's identification area and theexternal information area, said support (17′) also including kingpins(25) for attachment to the corresponding feeder.

FIG. 7 shows a plate support (17″) fitted with kingpins for attachmentto the feeder, in addition to a hole (26) for the lower lighting of theupper plate, and attachment flanges (27) for the plate itself.

Operation of the apparatus varies depending on whether the purpose is toanalyze the image of a panel with its biochemical reactions andantimicrobial susceptibility patterns occurring in different sections ofthe same panel, or the image of a plate with a colony growth and/orinhibiting halos.

In any case, prior to the reading session corresponding to the same typeof product, panel or plate, the apparatus must be calibrated afterselecting the support (17) to be used, which in turn is dependent on thepanel or plate to be processed.

Thus, once the support (17) is installed, which as previously statedmust be shaped as shown in FIG. 6, reference (17′), or shaped as shownin FIG. 7, reference (17″), or otherwise shaped depending on the type ofpanel or plate, prior to inserting a panel or plate, in order to allowthe light to pass freely through holes (22) or (26) through the support,the ‘calibrate’ option is executed from the control software residing ina personal computer and communicated with the apparatus through saidcommunications port (13). The camera then shows the histogram andreading values on the control personal computer screen, as shown in thegraph in FIG. 10, while indicator (4 b) is activated to indicate the endof the calibration reading.

If the calibration is within stable ranges, the process comes to an end;otherwise, chamber (2) adjustments are necessary.

The histogram provided by the system, as shown in FIG. 10, must have itsend peaks and valley as near as possible to the abscise axis.

The reading is performed as follows:

In the case of panels which, as previously stated, are polystyrenesupports fitted with a number of receptacles containing biochemicalsubstrates used for identifying a bacteria and others containing growthconcentrations of antimicrobial agents used in establishing a bacteria'scapacity to withstand the attack with the lowest possible antibioticconcentration—, the process starts by putting a reading algorithm towork which divides the panel's surface (28), shown in FIG. 5, into threesections corresponding to references (29), (30) and (31), with reference(29) corresponding to the type of panel identification area, reference(30) to the external information area and reference (31) to thereceptacles area.

In the first case, and as previously stated, area (29) identifies thetype of panel being used (three different types are available, accordingto microorganism, group of microorganisms or samples of microorganismsources). Area (29) is divided into four rectangles, although othergeometrical shapes are acceptable, printed preferably in the upperleft-hand section of panel (28) (for example, if the first and thirdreceptacles in area (29) are full, this would identify an enterobacteriapanel). Evidently, information on the type of panel is essential at thetime of interpreting the response of the bacteria when faced withcertain antibiotics.

Concerning area (30), foreseen for identifying external information usedin complementing the identification tests included in the panel, thearea comprises six squares, although other geometrical shapes areallowed, in which the user marks a point covering at least 90% of thesurface if the bacteria in question have reacted positively to aspecific laboratory technique (for example, a point covering the firstsquare of the upper portion would indicate that the microorganism hasreacted to the culture process at 18:00 hours as a result of an oxidasetest).

Concerning the receptacles zone or area (31), this area establishes theposition of each receptacle within the panel and calculates the panel'sreading area, generating four values for each receptacle.

As shown in FIG. 8, wherein reference (32) corresponds to the digitalcamera (15) focus, the receptacles (33) are cylindrical, the lower partor bottom (33′) thereof being conical in shape; also, the selection ofthe area to be considered in respect to the vertical of the digitalcamera fixedly installed in the upper part of the apparatus is essentialfor detecting possible bacterial growth. In said figure, references (34)correspond to the liquid culture medium and references (35) to thebacterial growth detection area, which are different in each casedepending on the location of the corresponding receptacle (33) inrespect to the vertical of the digital camera (32) focus.

Out of the four values generated by the system in the course ofprocessing a panel, the fist two are used for detecting bacterialmetabolism calorimetric reactions, corresponding to color code and colorsaturation, whereas the third and fourth values are used for detectingbacterial growth, corresponding to the growth area, and the amount oflight passing though each receptacle.

The values obtained for each receptacle (33) are then tabulated by thecontrol software in order to identify the microorganism causing theinfection and the best antibiotics against said infection, in theirlower concentration.

FIG. 9 shows a plate (36) in he form of a circular polystyrene support,which may acquire different shapes and sizes, containing a solid culturemedium on the surface of which is extended the microorganism suspensionpresumably causing the infection.

After inoculating the whole surface with the bacterial suspension, aselection of antibiotics-impregnated discs (37) is deposited thereon,the antibiotics contained in the discs then dissolving in and around theinoculated culture medium to achieve lower antibiotic concentrations inpoints further away from the edge of the disc. In said FIG. 9, the zoneunder reference (38) is the perimeter reconstruction zone resulting fromthe superposition of the ‘inhibiting halos’ from the two discs (37).

The capacity of the microorganisms to grow around an antibiotics disc,to a greater or lesser extent or none at all, determines theirclassification as sensitive or resistant to said antibiotic and thusestablishes the antibiotic's usefulness as an agent for fighting againstthe infection.

The microorganism forms a circular zone around each disc, referred toabove as an ‘inhibiting halo’, which is measured by the apparatus.

Thus, once the plate (36) is inserted in the chamber (2) and the orderis issued to process the image, a reading algorithm is activated whichdivides the surface into as many sections as discs are deposited on themedium. It then verifies that no halos are superposed, which is aphenomenon resulting from two discs located near each other generating ahalo of a diameter such that both overlap, so that, in the event of asuperposition, the system reconstructs the most probable halo for eachdisc, namely the previously mentioned zone (38).

The average diameter of each inhibiting halo is then calculated on thebasis of direct measurement of the diameters as such, or some otherparameter leading to this information. Three measurements are performedon each halo in order to calculate the average diameter, although adifferent number of measurements could likewise be implemented.

On completing the measurements, the system associates each measurementto a disc (37), according to its location on the plate (36), startingfrom the one on the upper portion and advancing clockwise.

The values obtained for each disc (37) are then tabulated by the controlsoftware in order to determine the antibiotics that are most convenientfor fighting against the microorganism causing the infection.

Obviously, in addition to the panel and plate readings according to theprevious description, a colony recount must be conducted, this naturallyconsisting in counting the number of bacterial groups that have managedto grow on the surface of a solid medium; according to the object of theinvention, said colony recount starts with a technique consisting in“sowing” a bacterial suspension on the surface of a medium contained ona plate, incubating it at 37° C. and establishing the number of groups(colonies) appearing after an 18- to 14-hour incubation period.

The system and the means associated thereto perform a color assessmentof the base medium on which the suspension was inoculated, the numericvalues ranging from 0 to 256.

In this respect, a colony is considered to be the smallest number ofadjacent pixels showing a 20% difference in color above or below thebase medium value.

Once the surface of a colony is established, said surface isextrapolated to all the areas presenting a 20% difference and acalculation is made as to how many times the surface of a colony iscontained in the overall surface; the value obtained is then tabulated,this operation being performed by the control software associated to thesystem in order to decide whether or not the number of colonies isindicative of the presence of an infectious process.

What is claimed is:
 1. A system for analyzing images produced bybacterial reactions, allowing microbiological reactions to be convertedinto numerical values which are subsequently used by a diagnosissoftware to determine: the amount of microorganisms present, indicativeof an infection; the identification of the microorganism present in thesample; and the infection-causing organism's antimicrobialsusceptibility pattern; wherein the reactions produced by the imageswhich are to be captured and interpreted by means of the system properoccur in liquid culture mediums contained inside panel receptacles or onthe surface of a solid culture medium or so-called plate, characteristicin that it comprises an apparatus (1) having a preferablyprismatic-rectangular shape which internally defines a closed hermeticchamber (2) internally fitted with a digital camera (15) mounted on asupport (16), a removable support (17) for the panel or plate concerned,and lighting means (18) and (19) complemented with light diffusers (20)and (21), whereas externally the apparatus (1), in correspondence withits front panel (3), includes a series of luminous indicators (4), afeeder (5) associated to the corresponding removable support (17), anopening button (6) acting on the feeder (5), a contrast switch (7) and ageneral switch (8); the rear panel (9) of said apparatus (1) beingdesigned to include a cover (10) providing access to the internalcomponents, as well as outlets (11), (12) and (13) supplying current tothe lighting fixture and to the chamber, and a port for communicatingwith the central computer, respectively, said rear panel (9) alsoincluding the connections (14) to a TDM 436 control module.
 2. A systemfor analyzing images produced by bacterial reactions, according to claim1, characteristic in that the support (17) is mounted in acoupling/uncoupling relationship with the feeder (5), the latter beingguided in its inward and outward movements to allow for thecorresponding receptacle panel (28) or plate (36) to be inserted.
 3. Asystem for analyzing images produced by bacterial reactions, accordingto claim 1, characteristic in that the support (17′) for the receptaclepanels (28) is shaped according to the shape of the panels, said supportbeing fitted with specific lateral recesses (23) and (24) and withkingpins (25) for attachment to the feeder (5), and also holes (22) forthe receptacles (33) of the corresponding panel (18).
 4. A system foranalyzing images produced by bacterial reactions, according to claim 1,characteristic in that the support (17″) for the plate (36) is fittedwith kingpins (25) for attachment to the feeder (5) and a hole (26) forthe disc that constitutes the plate (36), in addition to flanges (27)for attaching the plate.
 5. A system for analyzing images produced bybacterial reactions, according to claim 1, characteristic in that, onthe upper surface of panel (28), three areas (29), (30) and (31) areestablished, the first one for identifying the type of panel, the secondfor identifying the external information and the third for identifyingthe receptacle, with areas (29) and (30) corresponding to recesses (23)and (24) in the support (17).
 6. A system for analyzing images producedby bacterial reactions, according to claim 1, characteristic in that thesupport (17) fitted inside the chamber (2) of apparatus (1) is locatedabove the lighting means which constitute the fluorescent lights (18)and below the lighting means which constitute the fluorescent lights(19), the former being installed in the lower portion, whereas thelighting means or fluorescent light tubes (19) are located along thesides at an intermediate height, both being complemented by lightdiffusers (20) and (21), while the fluorescent light tubes (19) arecomplemented with metal plates (19′) which interrupt the trajectory ofthe light beam between the upper digital camera (15) and the panel oversupport (17).
 7. A system for analyzing images produced by bacterialreaction, according to claim 2, characteristic in that the support (17′)for the receptacle panels (28) is shaped according to the shape of thepanels, said support being fitted with specific lateral recesses (23)and (24) and with kingpins (25) for attachment to the feeder (5), andalso holes (22) for the receptacles (33) of the corresponding panel(18).
 8. A system for analyzing images produced by bacterial reactions,according to claim 2, characteristic in that the support (17″) for theplate (36) is fitted with kingpins (25) for attachment to the feeder (5)and a hole (26) for the disc that constitutes the plate (36), inaddition to flanges (27) for attaching the plate.
 9. A system foranalyzing images produced by bacterial reactions, according to claim 2,characteristic in that, on the upper surface of panel (28), three areas(29), (30) and (31) are established, the first one for identifying thetype of panel, the second for identifying the external information andthe third for identifying the receptacle, with areas (29) and (30)corresponding to recesses (23) and (24) in the support (17).
 10. Asystem for analyzing images produced by bacterial reactions, accordingto claim 3, characteristic in that, on the upper surface of panel (28),three areas (29), (30) and (31) are established, the first one foridentifying the type of panel, the second for identifying the externalinformation and the third for identifying the receptacle, with areas(29) and (30) corresponding to recesses (23) and (24) in the support(17).
 11. A system for analyzing images produced by bacterial reactions,according to claim 2, characteristic in that the support (17) fittedinside the chamber (2) of apparatus (1) is located above the lightingmeans which constitute the fluorescent lights (18) and below thelighting means which constitute the fluorescent lights (19), the formerbeing installed in the lower portion, whereas the lighting means orfluorescent light tubes (19) are located along the sides at anintermediate height, both being complemented by light diffusers (20) and(21), while the fluorescent light tubes (19) are complemented with metalplates (19′) which interrupt the trajectory of the light beam betweenthe upper digital camera (15) and the panel over support (17).
 12. Asystem for analyzing images produced by bacterial reactions, accordingto claim 3, characteristic in that the support (17) fitted inside thechamber (2) of apparatus (1) is located above the lighting means whichconstitute the fluorescent lights (18) and below the lighting meanswhich constitute the fluorescent lights (19), the former being installedin the lower portion, whereas the lighting means or fluorescent lighttubes (19) are located along the sides at an intermediate height, bothbeing complemented by light diffusers (20) and (21), while thefluorescent light tubes (19) are complemented with metal plates (19′)which interrupt the trajectory of the light beam between the upperdigital camera (15) and the panel over support (17).
 13. A system foranalyzing images produced by bacterial reactions, according to claim 4,characteristic in that the support (17) fitted inside the chamber (2) ofapparatus (1) is located above the lighting means which constitute thefluorescent lights (18) and below the lighting means which constitutethe fluorescent lights (19), the former being installed in the lowerportion, whereas the lighting means or fluorescent light tubes (19) arelocated along the sides at an intermediate height, both beingcomplemented by light diffusers (20) and (21), while the fluorescentlight tubes (19) are complemented with metal plates (19′) whichinterrupt the trajectory of the light beam between the upper digitalcamera (15) and the panel over support (17).
 14. A system for analyzingimages produced by bacterial reactions, according to claim 5,characteristic in that the support (17) fitted inside the chamber (2) ofapparatus (1) is located above the lighting means which constitute thefluorescent lights (18) and below the lighting means which constitutethe fluorescent lights (19), the former being installed in the lowerportion, whereas the lighting means or fluorescent light tubes (19) arelocated along the sides at an intermediate height, both beingcomplemented by light diffusers (20) and (21), while the fluorescentlight tubes (19) are complemented with metal plates (19′) whichinterrupt the trajectory of the light beam between the upper digitalcamera (15) and the panel over support (17).